Laboratory parameters related to disease severity and physical performance after reconvalescence of acute COVID-19 infection

Research into the molecular basis of disease trajectory and Long-COVID is important to get insights toward underlying pathophysiological processes. The objective of this study was to investigate inflammation-mediated changes of metabolism in patients with acute COVID-19 infection and throughout a one-year follow up period. The study enrolled 34 patients with moderate to severe COVID-19 infection admitted to the University Clinic of Innsbruck in early 2020. The dynamics of multiple laboratory parameters (including inflammatory markers [C-reactive protein (CRP), interleukin-6 (IL-6), neopterin] as well as amino acids [tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr)], and parameters of iron and vitamin B metabolism) was related to disease severity and patients’ physical performance. Also, symptom load during acute illness and at approximately 60 days (FU1), and one year after symptom onset (FU2) were monitored and related with changes of the investigated laboratory parameters: During acute infection many investigated laboratory parameters were elevated (e.g., inflammatory markers, ferritin, kynurenine, phenylalanine) and enhanced tryptophan catabolism and phenylalanine accumulation were found. At FU2 nearly all laboratory markers had declined back to reference ranges. However, kynurenine/tryptophan ratio (Kyn/Trp) and the phenylalanine/tyrosine ratio (Phe/Tyr) were still exceeding the 95th percentile of healthy controls in about two thirds of our cohort at FU2. Lower tryptophan concentrations were associated with B vitamin availability (during acute infection and at FU1), patients with lower vitamin B12 levels at FU1 had a prolonged and more severe impairment of their physical functioning ability. Patients who had fully recovered (ECOG 0) presented with higher concentrations of iron parameters (ferritin, hepcidin, transferrin) and amino acids (phenylalanine, tyrosine) at FU2 compared to patients with restricted ability to work. Persistent symptoms at FU2 were tendentially associated with IFN-γ related parameters. Women were affected by long-term symptoms more frequently. Conclusively, inflammation-mediated biochemical changes appear to be related to symptoms of patients with acute and Long Covid.


Laboratory analysis
At the first or second day of hospitalization, at FU1 and at FU2 the routine laboratory values were determined by the ISO 15,189 accredited Central Institute for Medical and Chemical Laboratory Diagnostic (ZIMCL) in Innsbruck, Austria as follows: CRP, IL-6, PCT, iron, transferrin, ferritin, folate, vitamin B12, creatine kinase (CK), troponin T, NT-proBNP, serum glutamic oxaloaxetic transaminase (GOT), serum glutamic pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) were analysed on a Cobas 8000 platform (Roche Diagnostics, Rotkreuz, Switzerland).Bioactive Hepcidin was determined by ELISA (DRG Instruments GmbH, Marburg, Germany) on a BEP2000, and 25-OH Vit D was measured by HPLC using the kit from Chromsystems Instruments and Chemicals GmbH (Graefelfing, Germany).All hematologic parameters were determined by an XN-2000 analyzer from Sysmex (Kobe, Japan).
Aromatic amino acids and the Trp derivative kynurenine (Kyn), as well as neopterin were analyzed at the Institute of Medical Biochemistry, Biocenter, Medical University of Innsbruck.The quantification of aromatic amino acids and Kyn as catabolite of Trp was performed as described previously 27 .Neopterin concentrations were measured by ELISA (BRAHMS Diagnostica, Berlin, Germany), nitrite was determined by Griess reaction 41 .The ratios of kynurenine/tryptophan (Kyn/Trp) and phenylalanine/tyrosine (Phe/Tyr) were calculated as a surrogate of IDO-1 42 and phenylalanine hydroxylase (PAH)-activity 43 .
All methods were performed in accordance with the relevant guidelines and regulations.

Questionnaire
The questionnaire, administered at multiple follow-up time points (FU1 and FU2) included questions about Eastern Cooperative Oncology (ECOG) score and sleep impairment.Acute COVID-19 infection data was collected retrospectively at FU1.The presence of sleep disturbance was determined either by the patient mentioning it or if there was prescription of sleep medication.

Additional data and definitions
Neurological symptoms were documented based on the patients' medical records.Fatigue data was gathered through the structured medical interview.COVID-19 severity were categorized based on the need for medical treatment, in accordance with the classification used by Sonnweber et al 44 .Mild severity was defined as outpatient treatment, moderate severity as inpatient treatment without respiratory support, severe disease as inpatient

Statistical analysis
Descriptive statistics include categorical variables, which were displayed as frequencies and percentages, and nonnormally distributed continuous variables, which were assessed using median and range.Most of the variables were not normal distributed; therefore, non-parametric tests were applied.Mann-Whitney U-test was used to compare two independent groups, Wilcoxon signed rank test yielded results for two dependent groups, and Kruskal-Wallis test was used for comparison of more than two independent groups.Post-Hoc tests for multiple comparisons included Bonferroni correction to counteract alpha error accumulation.Correlations were evaluated using Spearman rank test.The significance level was set to p < 0.05.All analyses were performed using IBM SPSS Statistics 28.0 (IBM Corp., USA).At FU1 12 patients reported an ECOG of 0 and 1 respectively, while an ECOG of 2 was reported in five patients and an ECOG of 4 in one patient.About one year after infection 14 patients had an ECOG of 0, 13 patients an ECOG of 1 and one patient an ECOG of 2. ECOG-Score was significantly higher during acute illness compared to FU1 (p < 0.001) and FU2 (p < 0.001).

Baseline characteristics
During the hospital stay for acute COVID-19 infection the percentage of men and women reporting symptoms like fatigue, sleep disturbance and neurological symptoms was comparable.However, on average, women were more frequently suffering from persisting symptoms in the reconvalescence phase (FU1 and FU2; see Table 1).62.5% of female patients were reporting neurological symptoms compared to 14.3% of men at FU2.This was surprising, as no woman had reported neurological symptoms at FU1. Sleep disturbance was prevalent in three out of four women, compared to 35% of male patients also at FU2. Sleep disturbance had already been more frequent in women at FU1 (62.5% women versus 27.3% of men).Moreover, half of the female patients were fatigued at FU2 and 23.8% of men.

Laboratory parameters
Several parameters differed significantly between acute COVID-19 infection and the two follow-ups (FU1-FU2) (see Table 2 for sex-stratified values).
Perturbations of IFN-γ mediated parameters between acute COVID-19 infection and FU1 have already been discussed in a previous publication 27 , therefore we herein only focus on FU2 results and dynamics: In the following only the most important findings will be summarized, the medians and p-values for all the following results are shown in Table 2 Regarding IFN-γ mediated parameters only few and rather slight changes between FU1 and FU2 were seen: In women, a further decrease at FU2 compared to FU1 was significant for Kyn/Trp.Interestingly, nitrite was elevated during acute infection and at FU1, while decreasing significantly between FU1 and FU2.
At the two follow-ups a high proportion of patients still had elevated levels of IFN-γ dependent laboratory values exceeding the 95th percentile of healthy blood donors (Geisler et al 41 ., see Table 3).Most notably, 69.6% of patients for Kyn/Trp and 65.2% of patients for Phe/Tyr at FU2 respectively had values above the 95th percentile.

Table 2. Sex-stratified study parameters and comparison of acute COVID-19 infection (acute) and FU1-FU2.
Median values and IQR of study parameters and longitudinal comparison between groups.*p value comparing parameters during acute COVID-19 infection (acute) and first follow-up (FU1); **p-value comparing parameters at FU1 and FU2; bold indicates significant differences; no significant difference (n.s.).white blood count (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH).B vitamin (folate and B12) availability, which was decreased at FU1 in men, improved from FU1 to FU2: Folate was significantly higher again in male patients at FU2 compared to FU1.Also, hemoglobin improved significantly in men at FU2 compared to FU1.
We observed a further reduction of ferritin values in male and female patients at FU2 compared to FU1.Other iron metabolism markers showed a return to baseline approximately within the first 60 days after symptom onset (FU1) and did not change afterwards, which is in line with previous observations on subjects with COVID-19 induced anemia 45 .
Inflammatory markers such as CRP and IL-6 were significantly higher during acute illness in both men and women compared with FU2.LDH was significantly elevated only in male patients during acute infection and declined to normal values until FU2.

Comparison of acute study parameters disease severity-stratified
We examined laboratory parameters during acute COVID-19 infection depending on disease severity.Again, the most important findings will be summarized, medians and p-values are shown in Table 4.
At FU1, vitamin B12, creatinine, WBC, MCH, platelet count, ANC, CRP, PCT and CK differed significantly among the three categories of disease severity (medians and p-values see Table 5).Neither tryptophan catabolism nor other IFN-γ mediated biochemical pathways differed according to the prior disease severity at FU1.However, about one year after symptom onset (at FU2), patients with a severe course of disease presented with significantly higher levels of Kyn/Trp than patients who had been admitted to the ICU during acute illness (40.3 vs. 28.9;p = 0.019).
The statistical comparison at FU2 showed higher levels for Phe and Tyr, as well as hepcidin25, MCH, and ferritin in patients with a lower ECOG score.LDH and transferrin showed inverse results (see medians and p-values in Table 6).www.nature.com/scientificreports/

Discussion
Since the 5th of May 2023 the World Health Organization no longer declares COVID-19 as a global health emergency.However, SARS-CoV-2 virus is endemic and will continue to challenge the public, as well as the scientific community.Research into the molecular basis of disease trajectory and Long-COVID is therefore essential.In view of that, we investigated IFN-γ induced parameters and routine laboratory values, as well as disease severity and ECOG score during acute COVID-19 infection and over the course of one year after symptom onset.As expected, ECOG performance status was significantly higher during acute COVID-19 infection compared to the two follow ups, indicating that acute infection strongly impaired physical performance.Affirmatively, this was widely reported in previous studies 7,46 .At the time of FU1 most patients had recovered a physical constitution within the range of ECOG 0-1, after one year about half of the patients were fully recovered and able to perform as before.Still, about half of the patients reported an impaired ability to work (ECOG 1) and presented with persistent symptoms after one year.
As metabolic changes induced by the virus and/or over-whelming immune response might be connected with the development of symptoms, we wanted to investigate the dynamics of different biochemical pathways, that have earlier been associated with symptoms like fatigue, depression or impaired quality of life.Furthermore, it was our goal to examine, whether disease severity influenced the investigated pathways long-term and whether patients with symptoms differed regarding the investigated lab parameters or not.
Numerous laboratory parameters showed notable differences between acute COVID-19 infection and follow-up investigations: Inflammatory parameters decreased within a standard range by the first follow-up, both in women and men.Interestingly, there appeared to be differences regarding immune response already during acute infection: Female patients presented with lower median concentrations of inflammatory markers CRP and IL-6 during acute illness, but higher neopterin values (also at FU1, at FU2 median concentrations of all the three inflammatory markers were within normal ranges in both men and women).In the past, the role of the X-chromosome among others was discussed as a possible reason for sex differences in immune regulation 47,48 .Male patients presented with low lymphocytes during acute infection, which steadily and significantly increased at FU1 and FU2, which is also consistent with earlier findings 7,49,50 .
Female patients were suffering more frequently than men from neurological symptoms, fatigue and sleep disturbance a year after infection which is supported by related research 51 .We cannot really explain the finding, that women did not report about neurological symptoms at FU1, while many reported them after 1 year: maybe deficits were regarded by women as mild and reversible initially, but in the course of reconvalescence they impaired everyday life strong.Another explanation might be that symptoms developed thereafter, which has also been stated by many other female patients who had a mild course of disease and later on developed symptoms of Long Covid.
Our data also fit well with results of an earlier study showing that men are more severely impacted in the acute phase, since mortality is higher in men 52 , and women are more likely to suffer long-term consequences.
In total, we observed a return to normal ranges regarding most investigated parameters by FU2, with negligible changes between FU1 and FU2 in our patient cohort.However, surprisingly, IFN-γ dependent laboratory values were still elevated one year after infection in a substantial number of patients.About two thirds of our cohort had values for Kyn/Trp (69.6%) and Phe/Tyr (65.2%) exceeding the 95th percentile compared to healthy adults.Thus, it seems that moderate to severe COVID-19 infection impairs IFN-γ mediated pathways up to one year after the onset of symptoms, which has not been previously reported and adds to our knowledge.
Moreover, there also appears to be a link between immune-mediated biochemical changes and clinical symptoms in our study.Patients, whose physical performance had returned to normal function, presented with higher concentrations of iron metabolism parameters (ferritin, transferrin, hepcidin) and amino acids (phenylalanine and tyrosine) than those with impaired ability to work and function (ECOG 1).Patients with sleeping problems tended to have higher Kyn and higher Kyn/Trp, while patients with fatigue had (rather unexpectedly) higher folate concentrations.Overall, the association of clinical symptoms with lab parameters influenced by IFN-γ signaling at FU2 appears to be more complex and individual, since patients with neurological symptoms at FU2 tended to have lower neopterin and Kyn concentrations.Also B vitamin metabolic changes appeared to be associated with tryptophan catabolism (both during acute infection and at FU) and disease severity: our data support a relationship between vitamin B12 and COVID-19 severity, which was already found in other studies 38,53 .Patients with a more severe disease and a continued higher impairment of their physical functioning ability had lower vitamin B12 levels at FU1 (and high MCV levels), indicating enhanced vitamin B12 demand during acute infection (probably as compensatory mechanism, reflected by high B12 levels in men) and lower availability during reconvalescent COVID-19 infection.Folate concentrations were interestingly also associated with tryptophan metabolism at FU1 and related with fatigue at FU1.Thus, our data indicate that metabolic changes are related with each other and with symptoms in reconvalescent patients.The small cohort size and a relative predominance of severe cases are major limitations which have to be taken into account and which restrict the generalizability of our results.On the other hand, the fact that the laboratory parameters were not influenced by immuno-suppressive treatment with e.g., corticoids (like in the later course of the pandemic) is a strength of this study, as immune-mediated biochemical changes could be monitored in a small, but well defined study population.Conclusively, similar investigations in larger cohorts of Long Covid patients are certainly needed to better extrapolate, whether and how exactly dysbalances of these biochemical pathways are really contributing to the persistence of symptoms in patients with Long Covid or not.
34 patients who had been hospitalized for COVID-19 infection came for FU1, 28 of them also for FU2 (one year after infection).The median age was 62 years (interquartile range (IQR) 54-71) ranging from 44 to 87 years.73.5% (male, n = 25) and 26.5% (female, n = 9).Three patients (8.8%) had a moderate severity, eleven patients (32.4%) had a severe disease, and 20 patients (58.8%) were admitted to the ICU during treatment for acute COVID-19 infection, representing a critical outcome.No patient in the study died because of SARS-CoV-2 infection.Median age was 60.0 years in the moderate group, 72.0 years in the severe group and 57.5 years in the ICU group.Regarding sex, statistical testing yielded no significant difference between men and women for disease severity (p = 0.561).During acute COVID-19 infection 19 out of the 34 patients of our sample reported an ECOG of 4, representing a total inability to care for themselves.Four patients reported an ECOG of 3, six patients reported an ECOG of 2, three patients reported an ECOG of 1, and one patient reported an ECOG of 0.

Table 1 .
. Frequencies of nominal study parameters during acute COVID-19 and at the two follow-ups.n, number of patients; missing, number of patients with missing value; valid %, percentage of patients excluding missing values; n = 34 during acute illness; n = 34 at FU1; n = 28 at FU2. Significant values are in [bold].

Table 3 .
41mber of patients exceeding the 95th or 5th percentile of IFN-γ dependent laboratory values during acute COVID-19 infection and at the two follow-ups (FU1, FU2).n, number of patients; valid %, percentage of patients excluding missing values; *according to Geisler et al41.; a 95th percentile; b 5th percentile.

Table 4 .
Comparison of study parameters during acute COVID-19 infection stratified by disease severity (only significant differences and statistical trends shown).Median values and IQR of blood values acquired during acute COVID-19 infection stratified by disease severity.Significant values are in [bold].*p-value comparing blood values among groups of disease severity.

Table 5 .
Comparison of study parameters at FU1 stratified by disease severity (only significant differences shown).Median values and IQR of blood values acquired at FU1 stratified by disease severity.*p-value comparing blood values among groups of disease severity.

Table 6 .
Comparison of blood values collected at FU2 ECOG-stratified (only significant differences and statistical trends shown).Median values and IQR of blood parameters collected at FU2 ECOG-stratified.*p-value comparing blood values among patients with an ECOG score of 0 and 1 at FU2, bold indicates significant differences; no significant difference (n.s.).